Abstract
Optical diffraction tomography (ODT) is a computational imaging technique based on refractive index (RI) contrast. Its application for microscopic imaging of weakly absorbing and scattering samples has been demonstrated by using a specially designed holographic microscope with angular scanning of the coherent sample illumination direction. Recently, an alternative low cost technique based on partially coherent sample illumination (PC-ODT), which is compatible with the conventional wide-field transmission microscope, has been established. In this case, the 3D refractive index distribution of the sample is obtained by deconvolution from a single stack of through-focus intensity images. The performance of PC-ODT has been successfully tested on various fixed specimens (diatom frustule and biological cells) and moving bacteria. Here, we demonstrate that the PC-ODT is an efficient tool for the analysis of living eukaryotic cell dynamics at short- and long-term periods. The COS-7 cells, which hail from the African green monkey kidney, have been chosen for this study. A fast data acquisition setup comprising an optical scanning module can be easily attached to the microscope, and it allows observing cell 3D organelle movements and RI variations, with the required temporal resolution. In particular, a more rapid nucleoli rotation than previously reported has been found. The long-term cell monitoring during necrosis reveals significant changes in cell dry mass concentration obtained from recovered RI contrast.
Highlights
While the theoretical fundamentals of the Optical diffraction tomography (ODT) were developed more than a half of century ago [1], its applications in high-resolution microscopy have been started relatively recently [2,3,4]
Data acquisition consists in angular scanning of sample illumination directions and the corresponding hologram recording
We shortly review the principle of the PC-ODT technique and describe the experimental setup used for its implementation
Summary
While the theoretical fundamentals of the ODT were developed more than a half of century ago [1], its applications in high-resolution microscopy have been started relatively recently [2,3,4]. PC-ODT Toward Practical Cell Study (487 nm) and axial (3.4 μm) resolutions are rather low for cell analysis Another technique which is based on angular scanning for non-interferometric microscopy has been reported [8], where Kramers–Kronig relations and oblique illuminations are exploited for phase recovery, instead of interferometric measurements. Let us consider that the sample satisfies the first-order Born approximation, which is suitable for weakly absorbing and low scattering samples Under this approximation, the 3D intensity distribution I(r) measured in a bright-field microscope (e.g., a stack of through-focus intensity images) can be written as the convolution of the point spread function (PSF), h (r), of the microscope and the sample’s scattering potential, as demonstrated in Ref [10,11,12,13,14]. The real part of the RI recovered from P(r) is considered for the cell dynamic analysis in Result and Discussions
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